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Preliminary observations on the in vitro photoreactivation of an insect virus inactivated with ultraviolet radiation
266
NOTES
two groups; those of the first group were reared individually and those of the second in groups of 50. Mortality was recorded every 24 hours in the individual rearings and every larva that died was examined microscopically. Periodic microscopic examinations were also carried out on live larvae taken from among those reared in groups of 50 to establish the characteristics and development of the disease for each test. Treatment at 100,000 rad resulted in a complete mortality after 10-12 days (Fig. 1, A and B) and at 300,000 rad after 48 hours. Doses of 10,000 and 50,000 rad seemed to have no effect on the larvae (Fig. 1, A and B). Mortality caused by virus was accelerated in virus-infected larvae irradiated with doses of 10,000 and 50,000 rad; these died after 11-12 days while nonirradiated infected larvae died after 14-16 days (Figs. 1, C,D,G, and H). However, at a dose of 100,000 rad the period of mortality was the same for larvae with and without virus infection, (Figs. 1, A,B,C, and D). Irradiation of dried polyhedra at 10,000 rad appears to have accelerated development of virus disease (Figs. 1, E and F); this was particularly evident for the third instar. Irradiation of the polyhedra at 300,000 and 500,000 rad did not noticeably influence mortality
Preliminary
Observations Insect
Virus
on the
Inactivated
It is well known that visible light can partially restore the activity of certain bacteriophages (R. Dulbecco, J. Bacterial., 59, 3294347, 1950) and plant viruses (F. C. Bawden and A. Kleczkowski, J. Gen. Microbiol., 8,145-l.%, 1953) which have been inactivated by suitable exposures to ultraviolet radiation ( UVR ) . Reactivation only
caused by virus; larval mortality was the same as for nonirradiated virus. It is noteworthy that irradiation of polyhedra at 800,006 rad reduced the effect of the virus, while irradiation at 2,000,OOO rad completely inactivated the polyhedra. In this last treatment no symptoms of the disease were found and morality did not differ from that of the controls. These observations suggest that: (1) irradiation with doses of 100,000 rad or more are lethal to third- and fourth-instar larvae of Malacosomu americanum; (2) exposure to radiations of 2,000,000 rad completely inactivates the polyhedra of the nuclear polyhedrosis of Malacosoma americanum; (3) development of the virus seems to be accelerated in larvae exposed to low doses of radiation. This last point should be further studied, especially by radiobiologists. ACKNOWLEDGMENT The author wishes to express his sincere thanks to Dr. J. Herman, Professor, Faculty of Sciences of Lava1 University, Quebec, Canada, for the use of the Gamma Cell 220.
W. A. SMIRNOFF
Forest Research Laborato y Sillery, Quebec, Canada Received January 5, 1967
in vitro
with
Photoreactivation
Ultraviolet
of an
Radiation
occurs in these cases when the bacteria or plants already infected with the UVirradiated virus are exposed to the visible light. It does not occur if the irradiated phage or virus, or the hosts or both, are exposed separately. It has been concluded that cellular structure is apparently essential for the occurrence of this phenomenon
267
iUOTES
(J. Kleczkowski and A. Kleczkowski, J. Gcn. Microbid., 8, 135-144, 1953). However, in the case of UV-inactivated Hemo$ilz~.s influenzae transforming DNA, cellular structure is not necessary (C. S. Rupert, J. GUI. Physiol., 43, 573-595, 1960)) and the same appears to hold for the insect virus rlsed in this investigation. When aliclnots were inactivated by exposure to UVR ( 250 or 260 nm ) and immediately exposed to strong visible light, the virus killed many more larvae, when fed to them, than did the virus in the control aliquots which had only received UVR. The virus used causes the well-known granulosis disease of the European cabbageworm (or the large white butterfly), Pieris bruxsicae. When exposed to the UVR it consisted of a pure suspension in distilled water of the intact inclusion bodies. This was prepared by repeated cycles of washing and centrifugation which included suspending the nearly purified virus twice
TBR1.E 1’11~
EFFECT
OF VI~IRLE
LIGHT
ox
THE
A~TITITY
on sucrose gradients. The 5% dilution of the concentrate exposed to the radiation contained about 4,120 X 10” + 400 X 10” capsules/ml. The exposures were made in a silica cell with a 10 mm square cross section using the monochromator, with its high pressure xenon arc source, which has been described by I. A. Magnus, A. D. Porter, K. J. McCree, J. D. Moreland, and W. D. Wright (Brit. 1. Dermatol., 71, 261266, 1959 ) . During the exposures the virus suspension was stirred by a fine stream of oxygen-free nitrogen bubbles. For feeding to the virus-free test larvae the virus suspension was diluted further ( see Table 1) and applied in measured quantities to standard cabbage leaves (W. A. L. David and B. 0. C. Gardiner, J. Invertebrate Pathol., 7,285290, 1965; 8,325333, 1966). The results obtained are summarized in Table 1. It can be seen that UV-irradiated virus which has been immediately exposed
1 OF T.LTRA~IoL~T-IRR~~IAT~I~
GR~TI-LOSIR
VIH~TS
.4vernge percentage kills of the test larvae
--IV:ivelength (nm)
Tltraviolet
irradiation -.
Intensity Ipa- /cm2)”
Total (pw-sec/cm2) _~~ __.
Visible light reactivation” ~_____._...
-2.50
560
5.3
x
101
Yes
-GO
560
5 3 x
101
X0
(‘orkrol -?tiO 260 260 Control
Dilutions the virus (7) 0.5 .-
at which was tested (94) 0.05
YX
X?
- -.~--__
P
-
23
76 68
1 ti 26
45 44;
11
0 1 O(I
--
2 100
loll -200
4 8 x 1 8 x
IO’ 10’
Yes
200
4 A x
10’
so
-
Yl3
I on
‘L The approximate irmdinnre of the I-YH at the front wall of the cell l~aserl on measurements with a Schwarz linear vacuum thermopile. b The visible light used for reactivation XIS also from the xenon arc pas?ed through R CPOR-n glass lense (minimum thickness 8 mm). It contained radiation of w:kvrlength ahove P&i nm, sOme long-wave TYlt, the whole visible spectrum, and some infrared radiation. The approximate intensity at the cell was 150,000 ~w/cm2 and the total exposure during reactivation approximately 400 X 106 rw-sec/cm2.
NOTES
268
to intense visible iight after UVR and before feeding to the larvae causes very significantly more deaths than UV-irradiated virus which has not been subsequently exposed to visible light. If the investigations which are now in progress confirm that reactivation has taken place, as seems likely, the results will demand a different explanation of the photoreactivation process in the case of this granulosis virus from that which has been
A Modification
accepted viruses.
for
phages and plant A. L. DAVID
W.
Agricultural Research Council Unit of Insect Physiology Cambridge, Engluncl I. A. MAGNUS Institute of Dermatology Homerton Grove, London, England Received Januuy
of Mallory’s Stain
for certain
Aniline
Oyster
Blue
18, 1967
Collagen
Tissue1
2. Remove mercury precipitate by placing in 70% alcoholic iodine solution for 10 minutes. 3. Wash in running water for 5 minutes. 4. Clear iodine in 5% sodium thiosulfate (hypo) solution. 5. Wash in running water for 10 minutes. 6. Stain in either Harris’ or Mayer’s hematoxylin for 20 minutes. 7. Rinse in tap water for 5 minutes. 8. Differentiate in 1% acid alcohol (3 Fixation to 5 quick dips) until nuclei are distinct Zenker’s solution gives excellent results against a light background. 9. Rinse in running water for 5 minutes. with oyster tissue. 10. Dip in ammonia water (3 drops of NH40H in 1000 ml of HSO) or saturated Technique lithium carbonate until sections are deep Embed in Paraplast and cut sections at blue. 6 P. 11. Rinse in running water for 5 minutes. 12. Stain in acid fuchsin solution, as outModified Procedure lined by Gridley (op. cit., 1960), for 7 1. Deparaffinize with xylene and pass minutes. slides through absolute and 95% alcohol 13. Rinse in tap water ( 2 quick dips ) . to water. 14. Blot to remove as much excess acid fuchsin from the slide as nossible without Mallory’s aniline blue collagen stain (Gridley, “Manual of Histologic and Special Staining Techniques,” 2nd ed., p. 60, McGraw-Hill, New York, 1960) was found to be a useful stain for oyster (Crasostrea gigas) tissue when the procedure was modified to eliminate overstaining. This note presents details on the modtied procedure that was used by Pauley and Sayce (I. Invertebrate Pathol., in press) to detect collagen in an oyster tumor.
1 This work is based on studies performed under contract No. 14-17-0007-441 between the Pacific Northwest Laboratory and the United States Department of Interior, Bureau of Commercial Fisheries.
touching
the
tissue,
1
15. Stain in aniline blue-orange G solution, as outlined by Gridley (op. cit., 1960), for 15 minutes.
NOTES
two groups; those of the first group were reared individually and those of the second in groups of 50. Mortality was recorded every 24 hours in the individual rearings and every larva that died was examined microscopically. Periodic microscopic examinations were also carried out on live larvae taken from among those reared in groups of 50 to establish the characteristics and development of the disease for each test. Treatment at 100,000 rad resulted in a complete mortality after 10-12 days (Fig. 1, A and B) and at 300,000 rad after 48 hours. Doses of 10,000 and 50,000 rad seemed to have no effect on the larvae (Fig. 1, A and B). Mortality caused by virus was accelerated in virus-infected larvae irradiated with doses of 10,000 and 50,000 rad; these died after 11-12 days while nonirradiated infected larvae died after 14-16 days (Figs. 1, C,D,G, and H). However, at a dose of 100,000 rad the period of mortality was the same for larvae with and without virus infection, (Figs. 1, A,B,C, and D). Irradiation of dried polyhedra at 10,000 rad appears to have accelerated development of virus disease (Figs. 1, E and F); this was particularly evident for the third instar. Irradiation of the polyhedra at 300,000 and 500,000 rad did not noticeably influence mortality
Preliminary
Observations Insect
Virus
on the
Inactivated
It is well known that visible light can partially restore the activity of certain bacteriophages (R. Dulbecco, J. Bacterial., 59, 3294347, 1950) and plant viruses (F. C. Bawden and A. Kleczkowski, J. Gen. Microbiol., 8,145-l.%, 1953) which have been inactivated by suitable exposures to ultraviolet radiation ( UVR ) . Reactivation only
caused by virus; larval mortality was the same as for nonirradiated virus. It is noteworthy that irradiation of polyhedra at 800,006 rad reduced the effect of the virus, while irradiation at 2,000,OOO rad completely inactivated the polyhedra. In this last treatment no symptoms of the disease were found and morality did not differ from that of the controls. These observations suggest that: (1) irradiation with doses of 100,000 rad or more are lethal to third- and fourth-instar larvae of Malacosomu americanum; (2) exposure to radiations of 2,000,000 rad completely inactivates the polyhedra of the nuclear polyhedrosis of Malacosoma americanum; (3) development of the virus seems to be accelerated in larvae exposed to low doses of radiation. This last point should be further studied, especially by radiobiologists. ACKNOWLEDGMENT The author wishes to express his sincere thanks to Dr. J. Herman, Professor, Faculty of Sciences of Lava1 University, Quebec, Canada, for the use of the Gamma Cell 220.
W. A. SMIRNOFF
Forest Research Laborato y Sillery, Quebec, Canada Received January 5, 1967
in vitro
with
Photoreactivation
Ultraviolet
of an
Radiation
occurs in these cases when the bacteria or plants already infected with the UVirradiated virus are exposed to the visible light. It does not occur if the irradiated phage or virus, or the hosts or both, are exposed separately. It has been concluded that cellular structure is apparently essential for the occurrence of this phenomenon
267
iUOTES
(J. Kleczkowski and A. Kleczkowski, J. Gcn. Microbid., 8, 135-144, 1953). However, in the case of UV-inactivated Hemo$ilz~.s influenzae transforming DNA, cellular structure is not necessary (C. S. Rupert, J. GUI. Physiol., 43, 573-595, 1960)) and the same appears to hold for the insect virus rlsed in this investigation. When aliclnots were inactivated by exposure to UVR ( 250 or 260 nm ) and immediately exposed to strong visible light, the virus killed many more larvae, when fed to them, than did the virus in the control aliquots which had only received UVR. The virus used causes the well-known granulosis disease of the European cabbageworm (or the large white butterfly), Pieris bruxsicae. When exposed to the UVR it consisted of a pure suspension in distilled water of the intact inclusion bodies. This was prepared by repeated cycles of washing and centrifugation which included suspending the nearly purified virus twice
TBR1.E 1’11~
EFFECT
OF VI~IRLE
LIGHT
ox
THE
A~TITITY
on sucrose gradients. The 5% dilution of the concentrate exposed to the radiation contained about 4,120 X 10” + 400 X 10” capsules/ml. The exposures were made in a silica cell with a 10 mm square cross section using the monochromator, with its high pressure xenon arc source, which has been described by I. A. Magnus, A. D. Porter, K. J. McCree, J. D. Moreland, and W. D. Wright (Brit. 1. Dermatol., 71, 261266, 1959 ) . During the exposures the virus suspension was stirred by a fine stream of oxygen-free nitrogen bubbles. For feeding to the virus-free test larvae the virus suspension was diluted further ( see Table 1) and applied in measured quantities to standard cabbage leaves (W. A. L. David and B. 0. C. Gardiner, J. Invertebrate Pathol., 7,285290, 1965; 8,325333, 1966). The results obtained are summarized in Table 1. It can be seen that UV-irradiated virus which has been immediately exposed
1 OF T.LTRA~IoL~T-IRR~~IAT~I~
GR~TI-LOSIR
VIH~TS
.4vernge percentage kills of the test larvae
--IV:ivelength (nm)
Tltraviolet
irradiation -.
Intensity Ipa- /cm2)”
Total (pw-sec/cm2) _~~ __.
Visible light reactivation” ~_____._...
-2.50
560
5.3
x
101
Yes
-GO
560
5 3 x
101
X0
(‘orkrol -?tiO 260 260 Control
Dilutions the virus (7) 0.5 .-
at which was tested (94) 0.05
YX
X?
- -.~--__
P
-
23
76 68
1 ti 26
45 44;
11
0 1 O(I
--
2 100
loll -200
4 8 x 1 8 x
IO’ 10’
Yes
200
4 A x
10’
so
-
Yl3
I on
‘L The approximate irmdinnre of the I-YH at the front wall of the cell l~aserl on measurements with a Schwarz linear vacuum thermopile. b The visible light used for reactivation XIS also from the xenon arc pas?ed through R CPOR-n glass lense (minimum thickness 8 mm). It contained radiation of w:kvrlength ahove P&i nm, sOme long-wave TYlt, the whole visible spectrum, and some infrared radiation. The approximate intensity at the cell was 150,000 ~w/cm2 and the total exposure during reactivation approximately 400 X 106 rw-sec/cm2.
NOTES
268
to intense visible iight after UVR and before feeding to the larvae causes very significantly more deaths than UV-irradiated virus which has not been subsequently exposed to visible light. If the investigations which are now in progress confirm that reactivation has taken place, as seems likely, the results will demand a different explanation of the photoreactivation process in the case of this granulosis virus from that which has been
A Modification
accepted viruses.
for
phages and plant A. L. DAVID
W.
Agricultural Research Council Unit of Insect Physiology Cambridge, Engluncl I. A. MAGNUS Institute of Dermatology Homerton Grove, London, England Received Januuy
of Mallory’s Stain
for certain
Aniline
Oyster
Blue
18, 1967
Collagen
Tissue1
2. Remove mercury precipitate by placing in 70% alcoholic iodine solution for 10 minutes. 3. Wash in running water for 5 minutes. 4. Clear iodine in 5% sodium thiosulfate (hypo) solution. 5. Wash in running water for 10 minutes. 6. Stain in either Harris’ or Mayer’s hematoxylin for 20 minutes. 7. Rinse in tap water for 5 minutes. 8. Differentiate in 1% acid alcohol (3 Fixation to 5 quick dips) until nuclei are distinct Zenker’s solution gives excellent results against a light background. 9. Rinse in running water for 5 minutes. with oyster tissue. 10. Dip in ammonia water (3 drops of NH40H in 1000 ml of HSO) or saturated Technique lithium carbonate until sections are deep Embed in Paraplast and cut sections at blue. 6 P. 11. Rinse in running water for 5 minutes. 12. Stain in acid fuchsin solution, as outModified Procedure lined by Gridley (op. cit., 1960), for 7 1. Deparaffinize with xylene and pass minutes. slides through absolute and 95% alcohol 13. Rinse in tap water ( 2 quick dips ) . to water. 14. Blot to remove as much excess acid fuchsin from the slide as nossible without Mallory’s aniline blue collagen stain (Gridley, “Manual of Histologic and Special Staining Techniques,” 2nd ed., p. 60, McGraw-Hill, New York, 1960) was found to be a useful stain for oyster (Crasostrea gigas) tissue when the procedure was modified to eliminate overstaining. This note presents details on the modtied procedure that was used by Pauley and Sayce (I. Invertebrate Pathol., in press) to detect collagen in an oyster tumor.
1 This work is based on studies performed under contract No. 14-17-0007-441 between the Pacific Northwest Laboratory and the United States Department of Interior, Bureau of Commercial Fisheries.
touching
the
tissue,
1
15. Stain in aniline blue-orange G solution, as outlined by Gridley (op. cit., 1960), for 15 minutes.